Engineering a CRISPRoff Platform to Modulate Expression of Myeloid Cell Leukemia (MCL-1) in Committed Oligodendrocyte Neural Precursor Cells

In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development. Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes. The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation. We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner. We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line. We validate the use of this tool by silencing MCL-1, an anti-apoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs. In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs). After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression. With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems. Key features • Generation of an inducible CRISPRoff Sleeping Beauty transposon system. • Experiments performed in vitro for generation of inducible CRISPRoff pluripotent stem cell line amenable to oligodendrocyte differentiation. • Strategy to downregulate an essential gene at different stages of oligodendrocyte development.

In vitro differentiation of human pluripotent stem cell (hPSC) model systems has furthered our understanding of human development.Techniques used to elucidate gene function during early development have encountered technical challenges, especially when targeting embryonic lethal genes.The introduction of CRISPRoff by Nuñez and collaborators provides an opportunity to heritably silence genes during long-term differentiation.We modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of embryonic lethal genes at different stages of differentiation in a stable manner.We provide instructions on how to generate sgRNA transposon vectors that can be used in combination with our CRISPRoff transposon vector and a stable hPSC line.We validate the use of this tool by silencing MCL-1, an antiapoptotic protein, which results in pre-implantation embryonic lethality in mice; this protein is necessary for oligodendrocyte and hematopoietic stem cell development and is required for the in vitro survival of hPSCs.In this protocol, we use an adapted version of the differentiation protocol published by Douvaras and Fossati (2015) to generate oligodendrocyte lineage cells from human embryonic stem cells (hESCs).After introduction of the CRISPRoff and sgRNAs transposon vectors in hESCs, we silence MCL-1 in committed oligodendrocyte neural precursor cells and describe methods to measure its expression.With the methods described here, users can design sgRNA transposon vectors targeting MCL-1 or other essential genes of interest to study human oligodendrocyte development or other differentiation protocols that use hPSC model systems.

Background
In vitro systems of differentiation have become useful tools to dissect the molecular mechanisms involved in cell fate transitions.The use of human pluripotent stem cells (PSCs) has revolutionized our understanding of early stages of human development that were previously inaccessible.Different gene-targeting techniques, such as RNAi, TALEN, Cas9 nuclease, and CRISPRi/a, are available to investigate the function of specific genes during development [1][2][3].Partial gene knockdowns in PSCs are commonly used to study the function of genes that would be lethal if completely knocked out.However, most of these tools have transient genetic modifications and their effectiveness is diminished during in vitro differentiation of PSCs.Nuñez et al. pioneered CRISPRoff, a programmable epigenetic memory writer protein that can heritably silence genes [4].This tool has greatly benefited the field, allowing developmental researchers to further understand the function of genes during long-term in vitro differentiations, as epigenetic memory is stable.We built on the CRISPRoff system by adding a Sleeping Beauty transposon system containing a reverse tetracycline-controlled transactivator (Figure 1A).Additionally, we designed a single-guide RNA (sgRNA) transposon plasmid that allows for efficient genomic integration (Figure 1B).With the CRISPRoff and sgRNA transposon vectors described here, we were able to develop a novel experimental approach to inducibly silence expression of MCL-1, an anti-apoptotic protein, in oligodendrocyte lineage cells.Deletion of Mcl-1 results in pre-implantation embryonic lethality [5].Consequently, investigating the function of MCL-1 during development has barriers as mouse embryos are not able to implant and PSCs undergo apoptosis once MCL-1 is knocked down or inhibited via small molecules [6,7].This technical barrier has halted further research on the function of MCL-1 during early human neurodevelopment.With CRISPRoff, we silenced expression of MCL-1 at different stages of neurodevelopment.In this protocol, we outline how this set of tools can be used to investigate the potential function of MCL-1 during development of oligodendrocyte lineage cells.We adapted this technology and linked it to an established oligodendrocyte generation protocol [8] to validate the use of these novel tools during differentiation of human PSCs.Moreover, we detail how to generate sgRNA transposon vectors for other genes of interest that can be adapted into alternative differentiation systems.There is a lack of literature that combines culturing of human oligodendrocyte lineage cells with epigenetic editing strategies.This protocol aims to bridge that gap by using a proof-of-principle experiment where MCL-1 is silenced at an early stage of differentiation.This protocol can be used to investigate an array of genes that cause lethality in PSCs or for studying gene function at different stages of oligodendrocyte development.

A. Cloning strategy
Sleeping Beauty transposon plasmids (Figure 1) were cloned using NEBuilder ® HiFi DNA Assembly Mastermix and are available from Addgene as noted in section Biological materials above.Addgene also maintains a table of gRNA sequences available from the depository, which users can mine to determine whether genes of interest have been effectively targeted in CRISPRi or CRISPRoff experiments (https://www.addgene.org/crispr/grnas/).It is useful to note, however, that effective sgRNAs for CRISPRi are typically localized to a narrow window downstream of the transcription start site (TSS), while CRISPRoffmediated gene repression displays a wide targeting window spanning a distance in excess of 2 kb from the TSS.Thus, candidate sgRNA sequences that are not predicted by tools referenced above may be empirically tested and validated.Critically, users will need to identify potential Cas9 target sites that contain a protospacer adjacent motif (PAM) for Spyo Cas9 (5′-NGG-3′).These sites can be identified using tools such as the UCSC Genome Browser (https://genome.ucsc.edu/).The sgRNA sequences used in this protocol can be found in Table S1.an agar plate supplemented with ampicillin or, alternatively, carbenicillin (100 mg/mL) and incubate overnight at 37 °C [11].c.Perform a colony PCR using bacterial colonies.We use a primer that binds the U6 promoter (5′ttcttgggtagtttgcagtttt-3′) as a forward primer and the anti-sense oligonucleotide as a reverse primer.Use the bacterial colony as template DNA, making sure to swirl the colony once in PCR mix before also swirling the same tip in a small volume (i.e., 100 µL) of LB broth for growth of positively identified colonies.d.Positively identified clones should be grown in overnight cultures in 3-5 mL of LB supplemented with ampicillin (100 μg/mL) on a shaking incubator at 37 °C.After 16 h in culture, plasmid DNA should be extracted from E. coli using a Miniprep Plasmid Purification kit.e. Sanger sequencing can be performed spanning BbsI cut sites to ensure successful assembly of the sgRNA transposon vector.Again, we typically use the U6 primer noted previously for Sanger sequencing.Alternatively, users may also elect to submit samples for full plasmid sequencing service.

D. Maintenance of human embryonic stem cells (hESCs)
H9 hESCs were maintained in mTeSR1 on Matrigel-coated plates.Change media daily and passage when needed.
2. Coat plates (1.5 mL for each well of a 6-well plate) overnight in 37 °C incubator.3. (Optional) Wash cells with 1 mL of gentle cell dissociation buffer.Aspirate.

4.
Incubate with 1 mL of gentle cell dissociation buffer for 3-5 min at room temperature (RT) (check on the microscope for efficient dissociation).Aspirate buffer.5. Add fresh mTeSR1, scrape gently with cell lifter, and resuspend the cells with a serological pipette such that they are in small clumps.6. Split cells 1:6 (if the well is > 80% confluent) onto 6-well plates for maintenance.7. Incubate cells at 37 °C and 5% CO2.

F. Antibiotic selection
1. Ensure that the appropriate puromycin concentration needed has been previously validated from an antibiotic kill curve for the cell type of interest.For the experiments outlined here, a puromycin concentration of 0.8 mg/mL was used for selection.2. The day after passaging, supplement mTeSR1 with the appropriate concentration of puromycin.3. Continue feeding daily with mTeSR1 supplemented with puromycin for 14 days.It is expected for the negative control cells that did not receive DNA during transfection to die within the first few days.Similarly, untransfected cells in the experimental wells should all die after 14 days.Surviving cells are expected to have integrated the CRISPRoff system into their genome to generate a stable cell line.4. From this point forward, media must be supplemented with a half dose of puromycin.5. Continue to maintain cells to prepare them for the second transfection.6.At this stage, cryopreservation is recommended, as this stable cell line can be used for future transfections with different sgRNA pools.3.After a 15-min incubation at RT, add the transfection mix into a well of a 12-well plate.4. Add 1 mL of Accutase per well (for cells in a 6-well plate) to detach hESCs.Incubate for 5 min at 37 °C. 5. Dilute the Accutase solution by adding 2 mL of DMEM/F12 medium.6. Use a cell lifter to remove cells from well and gently pipette the mixture 2-5 times with the p1000 pipette to fully dissociate the hESC cell colonies to single cells.7. Transfer the cells in a conical tube.8. Centrifuge the cells at 200× g for 4 min at RT. 9. Resuspend the cell pellet in 1 mL of mTeSR1 containing 10 µM Y27632 and count the cells with a hemocytometer.10.Dilute cell suspension to 1.5 × 10 6 cells/mL.11.Place 500 µL of this cell suspension to the well containing the transfection mix.12. Incubate cells overnight at 37 °C with 5% CO2.Studies here involved 5 µL of cDNA for qPCR. 5. QuantStudio 3 Real-Time PCR machine, SYBR Green master mix, and manufacturer's instructions were used to set up the assay.The primers used to measure gene expression of MCL1 are found in Table S2.

G. sgRNA transfection
Figure 4 contains results of the experimental validation in hESCs.

9 Published: Jan 05, 2024 C. sgRNA transposon plasmid design 1 .
Cite as: Gil, M. et al. (2024).Engineering a CRISPRoff Platform to Modulate Expression of Myeloid Cell Leukemia (MCL-1) in Committed Oligodendrocyte Neural Precursor Cells.Bio-protocol 14(1): e4913.DOI: 10.21769/BioProtoc.4913.Preparation of sgRNA backbone a. Perform BbsI digest and backbone dephosphorylation by combining ~1 µg of sgRNA transposon plasmid (Addgene #203359), 1 µL of BbsI endonuclease, 2 µL of rCutSmart Buffer, 1 µL of Quick CIP, and water to bring to a total final volume of 20 µL in a sterile tube.Place at 37 °C for 1-3 h.b.Perform PCR purification or gel extraction to purify the digested backbone.In our experience, a PCR purification is as the excised fragment is 22 bp long.2. Preparation of sgRNA insert a. Design and obtain oligos containing sgRNA sequence(s) for integration at the BbsI sites in the sgRNA transposon plasmid (Addgene #203359).Sense and anti-sense oligos should contain the appropriate overhang specific to the BbsI cut site to allow for successful ligation (Figure 1).Reconstitute oligos to 100 µM using ultrapure water.b.Place 4 µL of each of the 100 μM stock of sense and anti-sense oligos along with 4 µL of T4 DNA ligase buffer, 1 µL of PNK, and 27 µL of ultrapure water in a sterile tube and mix well.c.Phosphorylate and anneal oligos using the following protocol, which can be programmed into a thermocycler: 37 °C for 30 min 95 °C for 10 min 85 °C for 1 min 75 °C for 1 min 65 °C for 1 min 55 °C for 1 min 45 °C for 1 min 35 °C for 1 min 25 °C for 1 min d.After oligos have been phosphorylated and annealed, dilute 1:50 in ultrapure water and mix well.3. Ligation and verification a. Add 75 ng of purified backbone along with 1 µL of insert (diluted annealed/phosphorylated oligos), 2 µL of T4 DNA ligase buffer, 1 µL of T4 DNA ligase, and water to bring to a final volume of 20 µL, mixing well.Incubate at 16 °C for 1 h.b.Transform the reaction mixture into chemically competent E. coli, such as DH5α strain, spread onto

Figure 2
Figure 2 outlines the experimental procedures in sections E-I.

Figure 2 . 1 . 2 . 3 . 4 . 5 . 6 . 9 . 10 . 1 . 1 . 2 .
Figure 2. Generation of cell lines.Day 0: Transfect human embryonic stem cells (hESCs) with CRISPRoff transposon and Sleeping Beauty 100× transposase plasmids.Blue wells indicate no DNA negative control and pink wells indicate experimental wells with DNA.Day 4: Passage cells when they reach 70%-80% confluency.Day 5: Start antibiotic selection with puromycin.Blue wells with -indicate no DNA negative control without puromycin.Blue wells with + indicate no DNA negative control with puromycin.Pink wells with -indicate experimental wells with DNA and without puromycin.Pink wells with + indicate experimental wells with DNA and with puromycin.Day 20: Transfect hESCs with sgRNA pool and transpose plasmids.Day 24: Passage cells when they reach 70%-80% confluency.Day 25: Start antibiotic selection with hygromycin.Blue wells withindicate no DNA negative control without hygromycin.Blue wells with + indicate no DNA negative control with hygromycin.Pink wells with -indicate experimental wells with DNA and without hygromycin.Pink wells with + indicate experimental wells with DNA and with hygromycin.Day 40: Validate stem cell properties.SB = Sleeping Beauty

Day 1 1 . 2 .
Place 250 µL of mTeSR1 containing 1 µL of 10 mM Y27632 into a well of a Matrigel-coated 12-well plate.Prepare a mixture with 1 µg of the Sleeping Beauty transposase plasmid [pCMV(CAT)T7-SB100] and 1 µg of the sgRNA pool in 200 µL of Opti-MEM I reduced serum medium supplemented with 6 µL of TransIT-LT1.a. Make sure to include a non-targeting (NT) sgRNA pool in addition to the sgRNA pool targeting the gene of interest.b.Include a mixture without DNA (DNA negative control).

L.
DNase treatment of RNA This protocol was adapted from New England Biolabs DNase I Reaction protocol.1. Set up DNase I reaction on ice (Recipe 1).The volumes indicated on the Recipe are per sample.2. Incubate in the thermocycler at 37 °C for 10 min.3. Inactivate the DNase I by adding 0.5 µL of 0.1 M EDTA solution to the reaction mixture.4. Heat in the thermocycler for 10 min at 75 °C.5.The RNA sample is now ready to be used for reverse transcription.M. cDNA synthesis and quantitative RT-PCR This protocol was adapted from Thermo Fisher High-Capacity cDNA Reverse Transcription kit and SYBR Green reagent.1. Set reverse transcription master mix (Recipe 2).The volumes indicated on the Recipe are per sample.2. Add 10 µL of the master mix to 10 µL of RNA sample µg) in a PCR tube and mix well by vortexing.Briefly spin down tubes.3. Run samples in the thermal cycler using the following conditions: 25 °C for 10 min 37 °C for 120 min 85 °C for 5 min 4 °C hold 4. Final cDNA reaction is 20 µL.Dilute to 200 µL by adding 180 µL of nuclease-free H2O after reaction.